Artificial low-density lipoprotein carriers for transport of substances across the blood-brain barrier

ABSTRACT

This invention relates to a highly efficient artificial low-density lipoprotein (LDL) carrier system for the targeted delivery therapeutic agents across the blood-brain barrier (BBB). In particular, this invention relates to artificial LDL particles comprised of three lipid elements: phosphatidyl choline, fatty-acyl-cholesterol esters, and at least one apolipoprotein. The present invention further relates to compositions, methods and kits comprising artificial LDL particles for targeting drugs to and across the BBB for the prevention and treatment of brain diseases.

This application claims priority to U.S. Provisional Application Ser.No. 60/430,476 that was filed on Dec. 3, 2002. TECHNICAL FIELD OF THEINVENTION

This invention relates to the use of artificial low-density lipoprotein(LDL) particles of efficiently target and deliver substances across theblood-brain barrier (BBB). The invention further provides artificial LDLparticle compositions, methods, and kits for the prevention andtreatment of a variety of brain diseases.

BACKGROUND OF THE INVENTION

The blood-brain barrier (BBB), while providing effective protection tothe brain against circulating toxins, also creates major difficulties inthe pharmacological treatment of brain diseases such as Alzheimer'sdisease, Parkinson's disease, and brain cancer. Most charged molecules,and most molecules over 700 Daltons in size, are unable to pass throughthe barrier, and smaller molecules may be conjugated in the liver. Thesefactors create major difficulties in the pharmacological treatment ofdiseases of the brain and central nervous system (CNS), such asAlzheimer's disease, Parkinson's disease, bacterial and viral infectionsand cancer.

Many therapeutic agents for the treatment of diseases and disorders ofthe brain and CNS are sufficiently hydrophilic to preclude directtransport across the BBB. Furthermore, these drugs and agents aresusceptible to degradation in the blood and peripheral tissues thatincrease the dose necessary to achieve a therapeutically effective serumconcentration. The present invention provides a method of deliveringtherapeutic agents to the BBB by encapsulating the therapeutic agent inan artificial low-density lipoprotein particle (LDL). The LDL of thisinvention facilitates transport of therapeutic agents across the BBB bytranscytosis. Since most drugs and therapeutic agents that are toohydrophilic to cross the BBB are also too hydrophilic to be incorporatedinto an LDL particle, the present invention provides a method forproducing conjugates of the therapeutic agents with an LDL componentthat facilitates its incorporation into an LDL particles, transportacross the BBB and subsequent release of the therapeutic agent into thecell.

Prior methods for delivery drugs across the BBB involve three generalcategories: (1) liposome-based methods, where the therapeutic agent isencapsulated within the carrier; (2) synthetic polymer-based methods,where particles are created using synthetic polymers to achieveprecisely-defined size characteristics; and (3) direct conjugation of acarrier to a drug, where the therapeutic agent is covalently bound to acarrier such as insulin.

A. Liposomes

Liposomes are small particles that form spontaneously when phospholipidsare sonicated in aqueous solution, and consist of a symmetrical lipidbilayer configured as a hollow sphere surrounding an aqueousenvironment. This has appeal as a means of transporting water-solubledrugs through the cell membrane, as the phospholipid can be absorbed inthe plasma membrane, which automatically releases the contents of theliposomes into the cytosol. More successful variations of this techniqueinclude the use of cationic lipids, which can cooperatively createnanopores in the membrane. Cationic lipids are extensively used in cellculture to introduce water-soluble materials such as DNA molecules intocultured cells for experimentation.

Liposomes are attractive for transporting drugs across the BBB becauseof their large carrying capacity. However, liposomes are generally toolarge to effectively cross the BBB, are inherently unstable, and theirconstituent lipids are gradually lost by absorption by lipid-bindingproteins in the plasma. For example, in some studies, the large size ofthe liposomes used produced microembolisms that gave a false impressionof brain uptake. In some studies, liposomes were co-injected withPolysorbate 80, a detergent that can disrupt the BBB, as a stabilizingagent. The disruption of the BBB by the Polysorbate 80 in these studiesmay be responsible for any observed transport across the BBB.

Consequently, liposomes have had a checkered history as vehicles fortransporting drugs across the BBB. Several attempts have been made todirect the liposome to particular cellular targets. Peptidomimetic mAbsthat target endogenous receptors of the BBB have been used to targetpegylated immunoliposomes to various BBB receptors, with the aim ofachieving receptor-mediated uptake. However, this approach also requiresthe expensive production, testing and governmental approval ofmonoclonal antibodies. Because mAbs are typically produced in mice, andare susceptible to degradation, introduction of a peptidomimetic mAbwould not only face significant regulatory obstacles but would provedifficult to deploy in a patient environment.

Immunoliposomes, for example, have been constructed in a process thatinvolves covalent attachment of monoclonal antibodies (mAbs) to thesurface of the liposome. Since these immunoliposomes are immediatelycoated with plasma proteins that trigger uptake by thereticuloendothelial systems (RES), a system that avidly destroysmAb-conjugated liposomes, immunoliposomes have been treated withpolyethylene glycol (PEG) in a process known as pegylation.Unfortunately, the PEG molecules interfere with the mAb, rendering themnon-specific due to steric interference. Huwyler et al. (1996) Proc.Nat'l. Acad. Sci USA 93: 14164-14169 avoided this problem by creatingimmunoliposomes with a maleimide moiety at the tip of the PEG tail,which could be conjugated with a thiolated mAb. These pegylated OX26immunoliposomes, which were prepared with daunomycin in their interiors,were shown to be more stable in plasma than the free therapeutic agentor plain, unpegylated liposomes. Confocal microscopy, however, has shownthat although the liposomes were endocytosed into rat brain capillaries,they did not reach brain cells and remained attached to endothelialcells. Thus, pegylated and maleimide-treated liposomes appear to berelatively ineffective as drug delivery vehicles.

In 1997, Dehouck et al. discovered that the LDL receptors, which bindsApoE, is involved in transcytosis of LDL across the BBB. In a series ofthree publications, Versluis et al. described the use of ApoE-enrichedliposomes to deliver daunorubicin to cancer cells in mice. ApoE wasselected as an LDL-receptor targeting protein based on the finding thattumor cells express high levels of LDL receptors on their membranes.Versluis et al. (1998) also proposed using natural LDL, but thisexperiment was not attempted and subsequent papers focused exclusivelyon ApoE-enriched liposomes. Versluis et al. (1999) examined the tissuedistribution of daunorubicin, but there are no data related to brainuptake, indicating that this method was not envisaged as a means fortransporting daunorubicin across the BBB.

Additionally, the conjugation chemistry used by Versluis et al. isdifferent from that used in the present Invention. To anchor the drug tothe liposome membrane, the authors coupled 3α-O-(oleoyl)-4β-cholanicacid (an ester of lithocholic acid) to the tetrapeptide Ala-Leu-Ala-Leu,which was in turn covalently linked to the hydrophilic anti-tumor agentdaunorubicin. Thus, tumors were treated with conjugated, not free,daunorubicin. Although lithocholic acid is a steroid that alreadycontains an activatable acid group, the acid group is located on thesteroid side chain instead of the 3-OH position, which results in areaction product with less desirable features. Free daunorubicin can beproduced only after cleavage by proteases fund in the highly acidiclysosome, which would expose the conjugated during or agent todegradation by proteases, acid and other hydrolytic enzymes. Thetherapeutic agent would then be released into the intralysosomal spacewhere it could undergo further degradation and expulsion from the cell.

In contrast, the conjugates of the present invention preferably providesfor attachment of the therapeutic agent via an ester linkage that can beeasily cleaved in the cytosol and consequently escape the harshlysosomal conditions needed by the method of Versluis et al. Thus, atherapeutic agent conjugated by the present method would be more likelyto survive the journey to its target and to be released at the target inan efficient manner. It is also more likely to be transported across theBBB than a liposome.

The method of Versluis et al. also requires a large number ofsolid-phase peptide chemistry steps to synthesize the tetrapeptide, andseveral additional steps to conjugate it with FMOC and react theconjugate with lithocholic acid and finally with the drug. The presentinvention uses a much smaller number of steps, each of which producesnearly quantitative yield. Thus the present invention also offersimproved efficiency and lower cost.

Other liposomal formulations of doxyrubicin are currently in clinicaluse as possible treatments for cancer; however, no products have beenintroduced that use LDL.

Demeule et al. found that the protein melanotransferrin (p97) istransported by transcytosis across the BBB and concluded that an LDLreceptor was involved, suggesting that this protein be employed as adrug delivery system.

B. Synthetic Polymers

Synthetic polymers such as poly(butyl cyanoacrylate) or polyacrylamidecovered with Polysorbate 80 have also been tried. These polymers areappealing because the particles are sufficiently hydrophilic to bewater-soluble, yet are able to maintain their structural form for longperiods, which protects the therapeutic agent from uptake into the liverand kidney where it is subject to natural detoxification process. Inboth cases, uptake is generally supposed to occur by passive diffusionacross the cell membrane or as a defensive uptake by clathrin-coatedvesicles. In the former case, the therapeutic agent is then trapped inan endothelial cell, where it is not much closer to the target thanbefore, whereas in the latter case, the therapeutic agent is transportedto a lysosome, which is a highly acidic compartment in the cellcontaining proteases and other digestive enzymes analogous to stomachcontents. Thus, in the latter case, the therapeutic agent must remainstable throughout more extreme conditions. In neither case is the drugcarried across the cell and ejected into the brain parenchyma, which isthe desired result. Thus, it is not surprising that neither of these twomethods has achieved much clinical use.

Numerous researchers have tried various modifications of the approachesdescribed above to improve carrier uptake across the BBB with limitedsuccess. For example, Kreuter et al. (2002) J. Drug Target 10(4): 317-25engineered synthetic particles that contained various apolipoproteinsthat would bind to the apolipoprotein receptors located at the BBB. Theydemonstrated transport of drugs bound to poly(butyl cyanoacrylate)nanoparticles and coated them with Polysorbate 80. Uptake requiredcoating with Polysorbate 80, ApoE or ApoB. Apolipoproteins A11, C11, orJ coatings did not work. However, because these nanoparticles are notnaturally occurring, they may have undesirable side effects. Acrylatepolymers are particularly notorious for initiating autoimmune responses;the chemically-related polymer poly(acrylamide) is often used as anadjuvant.

Alyaudin et al. (2001) J. Drug Target 9(3): 209-21 usedpoly(butylcyanoacrylate) nanoparticles overcoated with Polysorbate 80 totransport [3H]-dalargin across the BBB and surmised the process was oneof endocytosis followed by possible transcytosis. This polymer may haveimmunological complications as well.

C. Therapeutic Agent Conougates

Direct conjugation of pharmacological agents with the substances thatcan be transported across the BBB, such as insulin, has also beenattempted. Insulin and insulin-like growth factors are known to crossthe blood brain barrier by specialized facilitated diffusion systems.(Reinhardt et al. (1994) Endocrinology 135(5): 1753-1761). Insulin istaken up by transcytosis mediated by the endothelial insulin receptor(Pardridge et al. (1986) Ann. Intern. Med. 105(1): 82-95). Specifictransporters also exist for glucose and for large amino acids such astryptophan. However, the specificity of the insulin transporter hasproved to be too high to allow pharmacological agents covalently linkedto insulin to cross into the brain. Similar results have been obtainedwith glucose and amino acid conjugates, whose uptake has been observedto obey the same general principles as other low-molecular weightsubstances, with only uncharged molecules below 700 Da achievingsignificant access to the brain. The inconvenience in devising chemicalsyntheses of conjugated forms of biomolecules, the risk of creatingunanticipated toxic effects, and the likely necessity of obtaining FDAapproval for an entirely novel compound has dampened enthusiasm for thisapproach.

Transport vectors, which are proteins such as cationized albumin, or theOX26 monoclonal antibody to the transferrin receptor undergoabsorptive-mediated and receptor-mediated transcytosis through the BBB,respectively. These have been used to transport small amounts of drug.This process, suffers from the high expense and difficulty of producingmonoclonal antibodies and cationized albumin and is not applicable toother types of molecules. Also, cationized proteins have been shown tobe toxic due to their immunogenicity and the formation of immunecomplexes that are deposited in the kidney.

Wu et al. (2002) J. Drug Target 10(3): 239-45 showed transport of humanbasic fibroblast growth factor (bFGF), a protein neuroprotective agent,across the BBB using a drug delivery vector consisting of a conjugate ofstreptavidin (SA) and the murine OX26 monoclonal antibody against therat transferrin receptor, and the conjugate of biotinylated bFGF(bio-bFGF) bound to a vector designated bio-bFGF/OX26-SA. Although theyshowed avid uptake of [¹²⁵I] labeled bio-bFGF into peripheral organs,only 0.01% of the injected dose was taken up per gram brain. Also, thisprocedure requires covalent modification of the drug, and may be usefulonly for limited classes of drugs. The carrier also contains mousemonoclonal antibodies as a component, which would cause an immuneresponse in the patient.

Kang et al. (2000) J. Drug Target 8(6): 425-34 also used anavidin-biotin linked chimeric peptide to transport a peptide across theBBB but achieved only 0.12% of the injected dose taken up per gram oftissue. Kang and Pardridge (Pharm. Res. 11: 1257-1264) conjugatedcationized human serum albumin with neutral light avidin, and then boundit to radiolabeled biotin. The biotin/cHSA/NLA complex was stable inblood for up to 24 h, but the conjugate was selectively degraded inbrain to release free biotin. As mentioned above, cationized proteinshave been shown to be toxic due to their immunogenicity.

Cationized monoclonal antibodies (mAbs) have also been used. Pardridge(J. Neurochem. 70: 1781-2) showed by confocal microscopy that the nativehumanized 4D5 MAb crossed the BBB by absorptive-mediated transcytosis,but only after cationization of the protein. This process, however,suffers from the high expense of producing and chemically modifyingmonoclonal antibodies and is not applicable to other types of molecules.

Witt et al. (2000) J. Pharmacol. Exp. Ther. 295(3): 972-8 used insulinto transport delta-opioid receptor-selective peptide D-penicillamine(DPDPE), a Met-enkephalin analog, across the BBB. Insulin, however,presents numerous hazards that limit its use as a therapeutic strategy.Also, other researchers have found the insulin receptor to be extremelyselective. Thus, in addition to the difficulty in producing chimericpeptides, this strategy is limited to a narrow class of pharmaceuticalagents.

Other researchers have attempted to conjugate drugs to glucose, forinstance using glycopeptides. However, no significant transport of anyglycopeptide via the BBB Glut1 transporter has ever been demonstrated.Attempts to use the high-transport rate of carrier-mediated transporterssuch as the Glut1 glucose transporter, the choline transporter, or theLAT1 large amino acid transporter have foundered on the problem thatcarrier transporters are too selective to accept conjugated substrates.They also suffer from the problem that p-glycoprotein, a member of themultidrug resistance gene, rapidly acts to actively remove many smallmolecules, including any drugs that manage to get across the barrier,from the brain.

In addition to the LDL receptor, the BBB also contains type II scavengerreceptors (SR), which bind LDL with high affinity. The scavengerreceptor is particularly good with modified forms of LDL such asacetylated LDL. Binding to the SR results only in endocytosis and notthe desired transcytosis. Rigotti et al. (1995) J. Biol. Chem. 270:16221-4 found that acetylated LDL is not transported across the BBB,whereas cationized bovine IgG was more effective Bickel et al. (1993)Adv. Drug. Del. Rev. 10: 205-245. The failure to demonstratetranscytosis with acetylated LDL discouraged many researchers fromattempting further experiments with LDL.

Protter et al. (WO 87/02061) describe a drug delivery system that usespeptides derived from apolipoproteins, such as ApoE and ApoB, which arecovalently attached to the pharmaceutical agent, or to a carriercontaining the agent. However, the use of molecular conjugates wouldonly be limited to a small number of drug classes, and subject to manyof the same problems discussed above.

Müller et al. (U.S. Pat. No. 6,288,040) describe the use of syntheticpoly(butyl cyanoacrylate) particles to which ApoE molecules arecovalently bound. The surface of the particles are further modified bysurfactants or covalent attachment of hydrophilic polymers. As statedabove, because these particles are not naturally occurring, they mayhave a variety of undesirable side effects.

Samain et al. (WO 92/21330) describe the use of synthetic particulatecarriers containing lipids that are covalently attached to a solid,hydrophilic core and that also contain ApoB for delivery of substancesto tumors or macrophages. However, they do not disclose any utility ofsuch vectors for delivering drugs across the BBB.

SUMMARY OF THE INVENTION

This invention relates to the use of artificial low-density lipoprotein(LDL) particles to target and deliver substances across the blood brainbarrier into the brain. Yet another object of the invention is thesynthesis of LDL carriers that are structurally stable, non-immunogenic,and protect a broad variety of drugs from degradation, inactivation,hydrolysis, conjugation, and uptake into non-target tissues. The presentinvention provides LDL particles to be used as carriers of therapeuticagents and improved methods of delivering such drugs and agents acrossthe BBB as compared to previously described methods. Unlike liposomes,for example, LDL particles are solid particles that consequently havegreater structural stability than liposomes. A further object of thepresent invention is to provide compositions, methods, and kitscomprising LDL carriers for the treatment and prevention of a broadvariety of brain diseases.

The present invention provides a process for conjugating hydrophilictherapeutic agents with cholesterol to facilitate incorporation of theconjugated therapeutic agent into an artificial LDL particle of thepresent invention. In a preferred embodiment, the present inventionprovides cholesterol-conjugated adriamycin and tetracycline. Theprocesses and resultant cholesterol conjugates and compositions of suchconjugates are useful in providing an LDL particle for the purpose oftransporting therapeutic agents across the brain barrier bytranscytosis, which is a receptor-mediated process that operates inbrain capillary endothelial cells as a means of importing cholesteroland essential fatty acids into the brain, that will facilitate andimprove treatment of a variety of diseases and disorders of the brainand CNS. Alternatively, the cholesterol conjugates of the presentinvention are useful for the delivery of the corresponding therapeuticagent across the BBB without incorporation of the conjugate into the LDLparticles of the present invention.

In a preferred embodiment, the cholesterol conjugates of the presentinvention are linked through an ester linkage that allows release of thetherapeutic agent from the conjugate by the action of ubiquitousendogenous esterases. Inclusion of the cholesterol conjugates of thepresent invention into an LDL particle of the present invention protectsthe cholesterol conjugates from hydrolysis by these esterases.

The present invention further provides artificial LDL particlescomprising egg yolk phosphatidyl choline (EYPC), cholesterol oleate, andapolipoprotein E3 (ApoE3). The components lipids form solid particlesthat contain three layers: a solid lipid core consisting of cholesterol,cholesterol esters, and an active agent; a middle interfacial layer,consisting of a mixture of fatty acid chains of phosphatidyl choline;and a surface layer, consisting of phospholipid head groups and ApoE3.

The LDL particles of the invention significantly increase the targetingof active agents to capillary endothelial cells and facilitate transportacross the blood brain junction by transcytosis. The protein andphospholipid components that surround the therapeutic agent also act toprotect it from degradation and uptake into non-target cells.

The present invention also relates to methods of treating diseases,ailments and conditions based upon the artificial LDLparticle-facilitated transfer of agents. For example, the presentinvention provides pharmaceutical compositions and methods for treatingvarious brain diseases comprising targeting specific agents to braintissues using the artificial LDL particles of the invention.

The present invention provides an artificial LDL particle comprising anouter phospholipid monolayer and a solid lipid core, wherein the outerphospholipid monolayer comprises at least one apolipoprotein and thesolid lipid core contains at least one therapeutic agent. In oneembodiment, the at least one apolipoprotein is selected from the groupconsisting of ApoA, ApoB, ApoC, ApoD, or ApoE, or an isoform of one ofthe apolipoproteins, or a combination of lipoproteins and/or isoforms.In a preferred embodiment, the at least one apolipoprotein is ApoE. In amore preferred embodiment, the at least one apolipoprotein is selectedfrom the group consisting of ApoE2, ApoE3 and ApoE4. The presentinvention also relates to artificial LDL particles further comprisingadditional targeting molecules or agents that enhance the targeteddelivery of the LDL complexes to brain tissue. In the most preferredembodiment, the at least one apolipoprotein is ApoE3, either alone or incombination with one or more oxysterols and/or an additionalapolipoprotein selected from the group consisting of ApoB and ApoE4.

The present invention provides an artificial LDL particle for thetransport of therapeutic agents across the blood-brain barrier. In apreferred embodiment, the at least one therapeutic agent is selectedfrom the group consisting of: amino acids, peptides, proteins,carbohydrates and lipids. In another embodiment, the at least onetherapeutic agent is a conjugate formed between cholesterol and an agentselected from the group consisting of: amino acids, peptides, proteins,carbohydrates and lipids. In preferred embodiments, the at least onetherapeutic agent is selected from the group consisting of: neurotrophicfactors, growth factors, enzymes, neurotransmitters, neuromodulators,antibiotics, antiviral agents, antifungal agents and chemotherapeuticagents.

The outer phospholipid monolayer of the artificial LDL particle maycomprise any phospholipid or combination of phospholipids, including butnot limited to phosphatidic acid, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, andthe like. In a preferred embodiment, the outer phospholipid monolayercomprises phosphatidylcholine and at least one apolipoprotein.

The artificial LDL particles of the present invention are particles witha preferred diameter between about 15 and 50 nm. In a more preferredembodiment, the artificial LDL particles have a diameter between about20 and 30 nm. The artificial LDL particles of the present invention havea preferred density between about 1.00 and 1.07 g/ml. In a morepreferred embodiment, the artificial LDL particles have a densitybetween about 1.02 and 1.06 g/ml. Furthermore, the artificial LDLparticles of the present invention have a serum stability of at leasttwo hours.

The present invention provides artificial LDL particles that aretransported across the BBB by transcytosis. In a preferred embodiment,the particles of the present invention have at least a 3-fold greateruptake specificity for brain compared to liver.

The solid lipid core of the artificial LDL particles of the presentinvention may comprise one or more lipids, including but not limited totriacylglycerols, cholesterol, cholesterol esters, fatty-acyl esters,and the like. In a preferred embodiment, the solid lipid core comprisescholesterol esters wherein the cholesterol is esterified with asaturated fatty acid, including but not limited to myristate, palmitate,stearate, arachidate, lignocerate, and the like, or an unsaturated fattyacid, including but not limited to palmitoleate, oleate, vaccenate,linoleate, linolenate, arachidonate, and the like. In a more preferredembodiment, the solid lipid core comprises the cholesterol estercholesterol oleate. In a preferred embodiment, the solid lipid core ofthe artificial LDL particles of the present invention comprise at leastone therapeutic agent that is a conjugate formed between cholesterol andadriamycin or tetracycline. In a preferred embodiment, the cholesteroland therapeutic agent of the conjugate are linked by an ester bond.

The present invention also provides compositions for delivery of atherapeutic agent across the blood-brain barrier comprising anartificial LDL particle of the present invention and a pharmaceuticallyacceptable carrier.

The present invention also provides a conjugate comprising cholesterollinked to a therapeutic agent selected from the group consisting of:amino acids, peptides, proteins, carbohydrates and lipids. In apreferred embodiment, the therapeutic agent is selected from the groupconsisting of: neurotrophic factors, growth factors, enzymes,neurotransmitters, neuromodulators, antibiotics, antiviral agents,antifungal agents and chemotherapeutic agents. In a more preferredembodiment, the therapeutic agent of the conjugates of the presentinvention are adriamycin or tetracycline. In the most preferredembodiment, the cholesterol and therapeutic agents of the conjugates ofthe present invention are linked by an ester bond. Each of theconjugates of the present invention may be combined with apharmaceutically acceptable carrier, as described herein, and used inany of the methods of drug delivery of the present invention.

The present invention also provides a method of producing an artificialLDL particle of the present invention comprising the steps of: 1)suspending phospholipids containing conjugated or unconjugatedtherapeutic agent in a buffer solution; 2) sonicating the solution toform the outer phospholipid monolayer and solid lipid core; and 3)adding a solution comprising at least one apolipoprotein, wherein theapolipoprotein is incorporated into the outer phospholipid monolayer. Ina preferred embodiment, the artificial LDL particles produced by themethods of the present invention have a diameter between 10 and 50 nm.

The present invention also provides a method for delivery a substanceacross the blood-brain barrier, said method comprising administering aneffective amount of any of the compositions of the present invention toa mammal in need thereof.

The present invention also provides kits for delivering substancesacross the blood-brain barrier, wherein said kits comprise a containercontaining the any of the compositions of the present invention andinstructions for use.

The present invention also provides for the use of the conjugates,artificial LDL particles and compositions of the present invention inthe production of a medicament for the treatment of diseases of thebrain and central nervous system (CNS), such as Alzheimer's disease,Parkinson's disease, bacterial and viral infections and cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Potassium bromide density gradient profile of radiolabeledlipids and LDL.

FIG. 2: Uptake of LDL and lipids in brain (left) and liver (right).

FIG. 3: (Left). Ratio of LDL uptake to lipid particle uptake in brainand liver. (Right). Ratio of brain uptake for LDL and lipid particles.

FIG. 4: Time course of plasma levels of ¹⁴C-Labeled LDL particles.

FIG. 5: Chemical structure of a hydrophilic molecule linked tocholesterol via a phthalate ester. The specific hydrophilic moleculeshown is adriamycin. Without conjugation, adriamycin alone is toohydrophilic and cannot be incorporated into LDL particles.

FIG. 6: Chemical structure of a hydrophilic molecule linked tocholesterol via a thioether ester. The specific hydrophilic moleculeshown is tetracycline. Without conjugation, tetracycline alone is toohydrophilic and cannot be incorporated into LDL particles.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term “artificial LDL particle” means a structurecomprising a spherical phospholipid monolayer and a solid lipid core.

As used herein, the term “liposome” means a structure comprising aspherical lipid bilayer and an aqueous core.

As used herein, the term “uptake specificity” refers to the ratio ofartificial LDL particle uptake to lipid particle (same as artificial LDLparticle except apoprotein is not included in outer phospholipidmonolayer) uptake in brain and liver. The uptake of artificial LDLparticles and lipid particles is measured in both brain and liver twohours after injection into Sprague-Dawley rats, as described herein. Theuptake specificity is calculated by dividing the ratio of artificial LDLparticle uptake to lipid particle uptake in brain by the ratio ofartificial LDL particle uptake to lipid particle uptake in liver.

As used herein, the term “serum stability” means the length of time atleast 75% of the injected artificial LDL particle remains in the plasma.

As used herein, the terms “apolipoprotein” and “apoprotein” mean aprotein associated with the phospholipid monolayer of lipoproteins,including but not limited to ApoA; ApoB; ApoC; ApoD; ApoE; and allisoforms of each.

As used herein, the term “ApoE” means one or more of the isoforms ofApoE, including but not limited to ApoE2, ApoE3 and ApoE4.

As used herein, the term “ApoB” means one or more of the isoforms ofApoB, including but not limited to ApoB48 and ApoB-100.

As used herein, the term “outer phospholipid monolayer” means amonolayer comprising at least one phospholipid where the phosphate headgroups of the phospholipids are oriented outwardly and the fatty-acylchains are oriented inwardly toward the solid lipid core. Phospholipidsinclude but are not limited to phosphatidic acid, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, andthe like.

As used herein, the term “solid core” means that portion of anartificial LDL particle enclosed by a spherical phospholipid monolayer.The solid core may comprise one or more lipids, including but notlimited to triacylglycerols, cholesterol, cholesterol esters, fatty-acylesters, and the like. As used herein, the term “cholesterol esters”refer to cholesterol esterified with a saturated fatty acid, includingbut not limited to myristate, palmitate, stearate, arachidate,lignocerate, and the like, or an unsaturated fatty acid, including butnot limited to palmitoleate, oleate, vaccenate, linoleate, linolenate,arachidonate, and the like.

As used herein, the term “therapeutic agent” means therapeuticallyuseful amino acids, peptides, proteins, nucleic acids, including but notlimited to polynucleotides, oligonucleotides, genes and the like,carbohydrates and lipids. The therapeutic agents of the presentinvention include neurotrophic factors, growth factors, enzymes,antibodies, neurotransmitters, neuromodulators, antibiotics, antiviralagents, antifungal agents and chemotherapeutic agents, and the like. Thetherapeutic agents of the present inventions include drugs, prodrugs andprecursors that can be activated when the therapeutic agent is deliveredto the target tissue.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition or compound with which an active ingredient may becombined and which, following the combination, can be used to administerthe active ingredient to a subject. As used herein, the term“physiologically acceptable” ester or salt means an ester or salt formof the active ingredient which is compatible with any other ingredientsof the pharmaceutical composition, which is not deleterious to thesubject to which the composition is to be administered. As used herein,“pharmaceutically acceptable carrier” also includes, but is not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; stabilizing agents; andpharmaceutically acceptable polymeric or hydrophobic materials. Other“additional ingredients” which may be included in the pharmaceuticalcompositions of the invention are known in the art and described, forexample in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa., which is incorporated herein by reference.

As used herein, “an effective amount” is an amount sufficient to producea therapeutic response.

Properties of LDL Particles and Apoproteins

The present invention relates to the discovery that artificial LDLparticles efficiently target and deliver substances across theblood-brain barrier by an active receptor-mediated process known astranscytosis. Transcytosis occurs naturally in brain capillaryendothelial cells as a means of importing cholesterol and essentialfatty acids into the brain. The artificial LDL carrier system of theinvention, therefore, provides a means of effectively targeting anddelivering drugs to the brain with minimal disruption of the BBB orother undesirable side effects.

Natural LDL particles have an average diameter of about 22 nm. The innercore consists of approximately 150-200 triglyceride molecules and1500-2000 cholesteryl ester molecules. The surface of the particlescontain a monolayer of about 450 phospholipid molecules, 185 moleculesof sphingomyelin, and a single molecule of apoprotein, typicallyApoB-100 (Hevonoja et al. (2000) Biochim Biophys Acta 1488(3): 189-210).Natural LDL particles may also contain about 600 molecules ofunesterified cholesterol and smaller amounts of lysophosphatidylcholine, phosphatidylethanolamine, diacylglycerol, ceramide, andphosphatidylinositol, as well as trace amounts of other biologicallipids (Hevonoja et al. (2000) Biochim Biophys Acta 1488(3): 189-210).Other apoproteins, including ApoE, are found in LDL, VLVL, and HDL, butpossess different receptor binding properties (Bradly et al. (1984) J.Biol. Chem. 259(23): 14728-35).

The surface of the LDL particle is, therefore, not uniformly coveredwith apoprotein, but consists of different regions with differentphysical properties. The apoprotein molecule is responsible formaintaining the structural integrity of the particle and for binding tolipoprotein receptors in the liver, kidney, and at the blood-brainbarrier. Apoproteins undergoes structural changes that depend on thestate of the lipid constituents (Mims et al. (1990) Biochemistry 29(28):6639-47).

Apoprotein E (ApoE) is a protein involved in cholesterol transport andplasma lipoprotein metabolism throughout the body. In peripheral cells,ApoE influences cellular concentrations of cholesterol by directing itstransport. In neurons, changes in cholesterol levels influence thephosphorylation status of the microtubule-associated protein tau at thesame sites that are altered in Alzheimer's disease. ApoE has three majorisoforms: ApoE4, ApoE3, and ApoE2, which differ by single amino acidsubstitutions. ApoE3 is the normal isoform, while ApoE4 and ApoE2 aredysfunctional. ApoE2 is associated with type-III hyperlipoproteinemia.ApoE4 is associated with increased risk for atherosclerosis andAlzheimer's disease, impaired cognitive function, and reduced neuriteoutgrowth. Except for age, ApoE4 is the most important risk factor insporadic Alzheimer's disease. ApoE4 may have toxic effects that dependon calcium (Veinbergs et al. (2002) J. Neurosci. Res. 67(3): 379-87),but its main effect appears to be to impair clearance of beta-amyloid byApoE3 (Holtzman et al. (2001) J. Mol. Neurosci. 17(2): 147-55). This hasbeen found to occur at the blood-brain barrier (Shibata et al. (2000) J.Clin. Invest. 106(12): 1489-99), and therefore could be an importanttherapeutic application.

Artificial LDL Particle Preparation

In a preferred embodiment, the artificial LDL particles of the inventioncomprise a mixture of egg yolk phosphatidyl choline (EYPC), cholesterololeate, and ApoE3. The component lipids form solid particles thatconsist of three layers (Hevonoja et al. (2000) Biochim. Biophys. Acta1488: 189-210): a solid lipid core containing cholesterol, cholesterolesters, and the active pharmacological agent, which can be eitherunconjugated or conjugated to the cholesterol; a middle interfaciallayer, containing a mixture of fatty acid chains of phosphatidylcholine; and a surface layer, containing phospholipid headgroups andApoE3.

The solid core and the presence of ApoE3, distinguishes the LDLparticles of the invention from liposomes, which consist of a sphericallipid bilayer surrounding an aqueous core and are unstable. In addition,the LDL particles are made of natural, non-immunogenic constituentswhich distinguishes them from artificial nanoparticles, molecular orchemical conjugates, or colloidal suspensions. The ApoE3 binds tospecific receptors on capillary endothelial cells that transport theentire particle across the junction by the active cell-mediated processof transcytosis. Once inside the brain, the therapeutic agent isnaturally released from the LDL particle as the cholesterol andphospholipids are gradually assimilated and utilized by the brain.

Although the lipid components stated above are preferred, this inventioncontemplates that other lipids, for example, LDL particles of differentlipid composition, including chemically-modified lipids, or admixturesof other naturally occurring lipophilic molecules could work equally aswell. One skilled in the art would understand that modifications may bemade to adapt the LDL carrier system for a specific therapeutic agent ortherapeutic application.

Preferably, the LDL particles are prepared with artificial LDL andcloned ApoE3. This greatly facilitates efficient and stableincorporation of the therapeutic agent into the lipid center of the LDL,and avoids problems with antigenicity due to possiblepost-translationally modified, variant, or impure ApoE3 protein purifiedfrom human donors. It also avoids possible inadvertent contamination ofthe ApoE3 or lipids with blood-borne diseases such as HIV or otherviruses. Such contamination is always a serious drawback of usinghuman-derived material.

In a preferred embodiment, the present invention relates to a modifiedmicroemulsion method of preparing artificial LDL particles comprisingthe steps of suspending the lipids, containing conjugated orunconjugated drug, in a phosphate buffered saline (PBS) solution, andsonicating the solution for 1 hour under a nitrogen atmosphere at 54° C.using a sonicator capable of delivering at least approximately 25 watts(18 μm amplitude of the probe at 22 kHz). This power level is importantfor creating LDL particles of an appropriate size to facilitatetransport across the BBB, preferably less than 50 nm in diameter, andmore preferably less than 30 nm in diameter. The sample containing theLDL particles is maintained at a constant temperature, preferablybetween about 53 and 56° C., by use of a water-jacketed sonicationchamber. Following sonication, the lipid solution is incubated with ApoEand the lipoprotein particles that are produced are separated byultracentrifugation in a potassium bromide (KBr) step gradient at285,000 g. The KBr is then removed by dialysis against PBS. Theparticles can be stored at 4° C. for later use, preferably up to twoweeks.

One skilled in the art would recognize that various aspects of themethod may be substituted. For example, other suitable densitygradients, such as cesium chloride or sucrose, may be substituted. In analternative embodiment, the LDL particles of the invention can beisolated by size exclusion chromatography, electrophoresis, or othermeans instead of centrifugation.

The method of preparation described herein produce drug-containing LDLparticles that are of an appropriate size to cross the BBB and maintainthe activity and structural stability of labile co-incorporatedmolecules. For delivery to the brain, the LDL particles should generallybe less than 50 nm in size, with a preferred range of 20 to 30 nm indiameter.

In another preferred embodiment, the LDL particles of the inventioncomprise a mixture of EYPC, cholesterol oleate, and ApoE3 present at aratio of 0.02 to 0.2 grams per gram of liposomes and provide efficientincorporation of the therapeutic agent and transcytosis across the BBB.In a more preferred embodiment, the range is 0.08 to 0.12 gram per gramof liposomes. More preferably, the molar ratio of EYPC to cholesterololeate to ApoE3 is 23:2.2 on a weight basis.

In yet a further embodiment of the invention, the LDL-based carriersystem may contain additional targeting molecules co-incorporated in thesurface layer to further facilitate transport and delivery of agents tothe brain. By way of example, oxidized derivatives of cholesterol(oxysterols) including cholesterol hydroperoxides, cholesterol epoxides,and hydroxycholesterol derivatives may also be used to improve uptake.The LDL particles may also incorporate other apoproteins such as ApoB orApoE4.

The present invention also relates to the incorporation of one or moreof a wide variety of substances including therapeutic agents to treat avariety of brain diseases or disorders. One of the advantages of the LDLcarrier system is the ability to deliver safety and naturally a broadvariety of drug classes, including those that are chemically unstable,highly reactive, or readily hydrolyzed in aqueous solution.

The list of possible agents include, but are not limited to:neurotrophic factors, such as NGF or the neurotrophic fragments producedfrom amyloid precursor protein, to treat brain injury andneurodegenerative diseases; enzymes, such as phenylalanine hydroxylase,to replace those lost through genetic defects; enzymes, such as tyrosinehydroxylase and DOPA decarboxylase, to regenerate dopamine that is lostin Parkinson's disease; enzyme activators or inhibitors to restore lostbiosynthetic function: antibiotics, such as tetracycline, for treatinginfectious diseases; neurotransmitters and neuromodulators for treatingpain, or conditions including disorders of movement, cognition, andbehavior; chemotherapeutic agents and anti-AIDS drugs, such asetoposide, ribavirin, or antihistamines, such as loratadine,fexofenadine, or certirizine (Zyrtec®), for treating brain tumors orother conditions with agents that do not reach the brain in sufficientamounts when tolerable doses are administered systemically; diagnosticagents, including contrast media like gadolinium derivatives, iohexol,or ioxaglate, or agents that are currently not used because of poorpenetration into the brain upon systemic administration; therapeutic ordiagnostic proteins such as antibodies, engineered and natural; andtherapeutic sequences encoding genes or proteins, or portions thereof,comprised of DNA, RNA, or amino acids that can be introducednon-invasively across the BBB.

The amount of therapeutic agent present in the LDL carrier will varywidely, depending on the type of molecule. For optimal incorporation inthe LDL carrier, the amount of therapeutic agent should be less than 0.1mol/mol cholesterol. It is expected that higher levels may destabilizethe LDL particles. This invention further contemplates that multipledrugs or additional agents may be present in the same particle.

In one embodiment, no covalent modification of the active substance isrequired for incorporation in the LDL particle of the invention,provided the substance is sufficiently hydrophobic to remain as amicroemulsion with cholesterol. This is true of most neutral andmoderately-charged molecules.

In another embodiment, if, for example, a therapeutic agent is highlycharged, like DNA or a peptide, or alternatively, to prevent diffusion,the agent can be covalently linked to cholesterol. In a preferredembodiment, the linkage is made with an ester bond, which would allowthe agent to be released by the ubiquitous esterases found in braintissue (Yordanov et al., (2002) J. Med. Chem. 45(11): 2283-8; Yang etal. (1995) Pharm. Res. 12(3): 329-36). Alternatively, one skilled in theart would be able to select other modes of attachment which would workequally as well, among them, illustratively, covalent attachment tophosphatidyl choline, some other lipophilic compound, or ApoE itself.

As state above, the LDL particles of the invention have severaladvantages when compared to other carriers such as pegylated liposomes,nanoparticles, and similar artificial substances. The LDL particles ofthe invention are made of normal constituents of blood plasma, bind totheir natural receptor, and are transported by normal pathways into thecells as part of the brain's natural requirement for exogenous essentialfatty acids. Therefore, their toxicity is significantly lower thanpolymer-based carriers and agents that disrupt cell membrane integrity.A current method used for delivering drugs to the brain is osmoticdisruption of the BBB with matnitol. Disrupting the BBB, however, hasserious drawbacks, among them the fact that it allows toxins and evenviruses to enter the brain along with the desirable therapeutic agentwhich can have serious collateral effects.

The artificial LDL carrier system of the invention also provide theadvantage that they remain at high levels in plasma for at least twohours (FIG. 4). This is important in maintaining a sufficient effectiveplasma concentration (often referred to as “area under the plasmaconcentration curve” or AUC) for adequate uptake and delivery ofsubstances to the brain.

By specifically targeting brain tissue, as shown in Example 3, theartificial LDL particles of the invention significantly increase thetherapeutic efficacy of drugs, because the drugs are much less likely tobe taken up by the liver and inactivated by detoxification pathways,including inactivation by liver enzymes such as P450.

Compositions Comprising Artificial LDL Particles

The LDL particles of the invention may be formulated in a variety ofways depending on the type of brain disease to be treated. The inventiontherefore includes within its scope pharmaceutical compositionscomprising at least one drug-containing LDL particle complex formulatedfor use in human or veterinary medicine. Such compositions may bepresented for use with pharmaceutically-acceptable carriers orexcipients, optionally with supplementary medicinal agents. Conventionalcarriers can also be used with the present invention. Acceptablecarriers include, but are not limited to, glucose, saline, and phosphatebuffered saline.

Following treatment to remove free therapeutic agent or othernon-incorporated molecules, the LDL particle suspension is brought to adesired concentration in a pharmaceutically acceptable carrier foradministration to a patient. Because the LDL particles are too large tobe efficiently absorbed parenterally, compositions are intended forintravenous use, but conceivably may also be administeredintramuscularly or intraarterially, or even parenterally or orally givenappropriate modifications.

Thus, this invention provides compositions for administration targetedto the BBB which comprise a solution of a selected active agentcontained in an LDL particle and a pharmaceutically acceptable carrier,preferably an aqueous carrier. The resulting compositions may besterilized and packaged for use in a kit, or filtered under asepticconditions and lyophilized. Kits for intravenous administrationcontaining a lyophilized preparation may also include a sterile aqueoussolution for mixing prior to administration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents and thelike, for example, sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, etc.

The concentration of the LDL particles in these formulations can varywidely, i.e. from less than about 0.5%, usually at or at least about 1%,to as much as 5 to 10% by weight. Additional methods for preparingintravenously administrable, LDL particle formulations will be known tothose skilled in the art. Such methods are described in detail in, forexample, Goodman & Gilman, The Pharmacological Basis of Therapeutics byJoel G. Hardman (Editor), Lee E. Limbird McGraw-Hill Professional; ISBN:0071354697; 10^(th) edition (Aug. 13, 2001).

Therapeutic Methods of Using Artificial LDL Carriers

In a further embodiment, the artificial LDL particles may beadministered to a mammalian host in need of treatment to effectivelydeliver agents across the BBB to the brain. For use in therapy, aneffective amount of drug-containing LDL particles can be administered toa subject by any mode allowing LDL particles to be taken up by thecapillary endothelial cells.

In clinical applications, the LDL particle delivery system significantlyenhances the therapeutic efficacy of drugs for uses such as thetreatment of Alzheimer's disease, Parkinson's disease, and brain cancer.For example, a neurotrophic factor such as nerve growth factor could beincorporated into the LDL particles, enabling the peptide to be taken upinto the brain. This would cause the growth of new nerve processes thatcould be beneficial in a number of neurodegenerative diseases. Asdescribed herein, those skilled in the art would recognize that a broadvariety of alternative clinical applications exist using the LDL carriersystem of the invention.

Cholesterol-Conjugates of Therapeutic Agents.

Cholesterol is a relatively chemically-inactive molecule. Consequently,cholesterol must be activated prior to reacting the cholesterol with anamine-containing therapeutic agent. For example, cholesterol may beactivated by a reaction with a cyclic anhydride such as phthalic orsuccinic anhydride that produces a phthalate or succinate ester whichcontains a carboxyl group. The carboxyl group is then activated byreaction with pentafluorophenol and then reacted withdiisopropylcarbodiimide to create an amide linkage with anamine-containing therapeutic agent. The resulting product is acholesterol-therapeutic agent conjugate wherein the therapeutic agent isconjugated to cholesterol through an ester linkage. Thecholesterol-therapeutic agent conjugate is sufficiently lipophilic toallow incorporation into the artificial LDL particles of the presentinvention. Also, since the preferred linkage is an ester bond, thetherapeutic agent can be released from the cholesterol moiety by theaction of ubiquitous endogenous esterases. Thus, the release of thetherapeutic agent from the conjugate is not dependent on the harshconditions and action of non-specific peptidases found in lysosomes.Although the preferred linkage between cholesterol and a therapeuticagent is an ester bond, the present invention contemplates otherlinkages, including but not limited to ether, amide and peptide bonds.

Alternatively, an amine- or hydroxyl-containing therapeutic agent may beconjugated to thiocholesterol by reaction of thiocholesterol, atherapeutic agent and a bifunctional cross-linking reagent such as, butnot limited to, PMPI. The resultant conjugate is a conjugate ofcholesterol and a therapeutic agent linked by a thioether linkage. Ifthe therapeutic agent contains an amino group, it may be conjugated tothiocholesterol using one of the many commercially-availablebifunctional crosslinking agents, including but not limited tosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.

Once formed, the cholesterol-therapeutic agent conjugates may be mixedwith unconjugated cholesterol oleate, phospholipids, lipoproteins, asdescribed herein, thereby producing the artificial LDL particles of thepresent invention.

Each reference cited above is incorporated herein by reference, in itsentirety.

EXAMPLES

The following Examples serve to illustrate further the present inventionand are not to be construed as limiting its scope in any way.

Example 1 Purification of Full-Length Apoprotein E (ApoE)

DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) from Avanti PolarLipids, Inc. is suspended in benzene at a concentration of 100 mg/ml ina glass tube and sonicated using bench top sonicator. The DMPCsuspension is shell-frozen and lyophilized overnight, then resuspendedin 30 ml standard buffer (10 mM Tris-HCl. pH 7.6. 0.15M NaCl, 1 mM EDTA.0.0005% NaN₃) to make 10-20 mg/ml DMPC and transferred to a plastic 50ml conical tube. The tube is placed in a water bath and sonicated usinga sonicator with a microtip for four 10 min intervals, interspersed with2-3 min cooling. Sonication is repeated until the solution becomesnearly transparent. The sonicated DMPC is then centrifuged at 2000 rpmfor 20 minutes to remove any titanium that may have sloughed off duringsonification.

Bacterial cells expressing cloned ApoE are collected by centrifugationand sonicated on ice in a Branson Sonifier with a large tip, using fourperiods of high intensity sonification for 30 sec separated by 2 mincooling intervals. The sonicated suspensions are centrifuged at 39.000×gfor 20 min at 4° C. to remove cell debris and the supernatant iscombined with DMPC at 100 mg per liter of original culture medium. Themixture is incubated in a water bath at 24° C. (which is the transitiontemperature for DMPC) overnight.

The density of the DMPC-ApoE mixture is then raised to 1.21 g/ml byaddition of solid KBr and centrifuged in a step gradient (1.21, 1.10,and 1.006 g/ml) overnight at 55,000 rpm in a 60 Ti Beckman fixed-anglerotor at 15° C. The white band near the top of the tube containing freeDMPC is discarded, and the ApoE-DMPC complex below it, floating atdensity of 1.009 g/ml, is removed. The presence of ApoE is confirmed bygel electrophoresis. Appropriate fractions are then dialyzed against0.1M NH₄HCO₃ and 0.0005% NaN₃ at 4° C. against three changes of buffer.

The protein is then digested with thrombin to remove the polyhistidinetag left by the vector. Activated thrombin is mixed with the protein ata ratio of 1:500 thrombin:ApoE, w:w, and incubated at 37° C. for atleast 1 hr and aliquots are analyzed by gel electrophoresis to ensurethat the protein is completely cleaved. Because of the presence of twocleavage sites, incomplete cleavage would result in a second band with ahigher molecular weight. Once complete cleavage has been demonstrated,beta-mercaptoethanol is added to a 1% final concentration to stop thereaction. The ApoE is then shell frozen ApoE in a 50 ml acid washedtubes and lyophilized overnight.

The ApoE is then washed with 30 ml cold chloroform/methanol (2:1 v/v)and collected by centrifugation at 1500 rpm in a J6 Beckman centrifugeat 4° C. for 10 min. The pellet is resuspended in a small volume of coldmethanol and vortexed, and the tube is then filled with cold methanoland centrifuged. This step removes any remaining chloroform. Themethanol is poured off, leaving a small amount in the tube which isvortexed to make a paste out of pellet. Five ml of a solution containing6M guanidine-HCl. 0.1M Tris HCl, pH 7.4, 0.01% EDTA, 1%beta-mercaptoethanol, and 0005% sodium azide, is added and the solutionis loaded onto a Sephacryl S-300 column that has been equilibrated with4M guanidine-HCl, 0.1M Tris-HCl, pH 7.4, 0.1% beta-mercaptoethanol, and0.0005% NaN₃. The protein is eluted with 4M guanidine buffer thatcontains 0.1% beta-mercaptoethanol and 0.0005% sodium azide. Fractionsare dialyzed against 0.1M NH₄HCO₃ and 0.0005% NaN₃, with four changes ofbuffer. The purified ApoE is concentrated using YM10 Amicon Centriprepconcentrators (Millipore) to a final concentration of 1-2 mg/ml, andstored at −20° C.

Example 2 ApoE Enrichment of LDL-Liposomes

1. Preparation of lipid cake: Egg yolk phosphatidylcholine (25 mg) andcholesteryl oleate (2 mg) are dissolved (ratio 25:1) inmethanol/chloroform (2:3). The solvent is evaporated under nitrogen at4° C.

2. Preparation of liposomes: The lipid cake is hydrated in 12 ml of 10mM Tris-HCl buffer, p H 8.0, containing 0.1M potassium chloridepreviously bubbled under nitrogen gas. The mixture is sonicated for 1 hat 18-um output under nitrogen stream at 54° C. and centrifuge to removeany titanium particles produced during sonication.

3. Preparation of artificial LDL: The liposomes prepared in step 2 areincubated with ApoE protein in a ratio of 1/10 (w/w) protein/lipid for30 min at 37° C. The liposomes are then purified and concentrated bydensity gradient ultracentrifugation at 285,000×g for 18 h at 4° C.using a three-layer KCl step gradient with densities of 1.064, 1.019,and 1.006 g/cm³. KBr is added to the liposome solution to raise itsdensity to 1.21 and applied to the bottom of the centrifuge tube.Optiseal tubes (Beckman) are suitable for the ultracentrifugation step.After centrifugation, liposomes are visible as a narrow opalescent layerapproximately ¼ of the distance from the top of the tube. This layer isremoved and dialyzed overnight at 4° C. against PBS (phosphate-bufferedsaline) containing 1 mM EDTA. The LDL suspension is stable for at least7 and can be stored at −20° C. under argon or nitrogen.

Example 3 Uptake of Artificial LDL Particles in Brain

The ¹⁴C-LDL suspension in PBS, containing approx. 1 mg lipid, wasinjected into the tail vein of a Sprague-Dawley rat (150 g). At variousintervals, blood samples were obtained by cardiac puncture usingsyringes containing EGTA. Blood plasma, RBCs, and tissues werehomogenized in water and the ¹⁴C was measured in a scintillationcounter.

FIG. 2 shows the results of uptake of LDL and lipids in brain (left) andliver (right). Male Sprague-Dawley rats were injected intravenously withLDL or lipid particles containing ¹⁴C-cholesterol and radioactivity inbrain and liver was measured two hours later. Brain and liver took up19.8 and 4.7 times more label, respectively, from LDL compared to lipidparticles of identical composition except for the presence of ApoE. Thisindicates that the uptake was caused by transcytosis mediated by the LDLreceptor (p(>T)=0.00055).

FIG. 3 (left) shows the ratio of LDL uptake to lipid particle uptake inbrain and liver. Brain incorporated a higher percentage of LDL versuslipid particles compared to liver (p(>T)=0.0003) suggesting a 4-foldgreater specificity of LDL for the target organ compared to liver. FIG.3 (right) shows the ratio of brain uptake to liver uptake for LDL andlipid particles. The brain:liver ratio, another measure of organspecificity, was higher for LDL than for lipid particles,(p(>T)=0.0003). In other words, 5.88 times more lipid particles weretaken up by the liver than brain, while only 1.36 times more LDLparticles were taken up by the liver than brain (p(>T)=0.034).

FIG. 4 shows the time course of plasma levels of ¹⁴C-labeled LDLparticles. Blood ¹⁴C remained constant for at least 2 hours afterinjection of LDL, indicating that the label was not being removed fromcirculation by other organs.

Example 4 Conjugation of Primary Amines with ¹⁴C Cholesterol

Preparation of cholesterol-phthalate ester: Cholesterol (1 mg) isevaporated with nitrogen and lyophilized to remove ethanol. The solid isdissolved in a minimum volume THF and transferred to a glass reactionvial. Solid phthalic anhydride (1-2 mg, >4 equiv.) is added, followed by50 Et₃N. The mixture is heated at 1000 for 5 min and ≈20 μl pyridine isadded to make suspension clear. The mixture is heated at 100° for 30 minuntil solution is red and purified on TLC (Silica/UV254 plates) withEtOH/toluene (2:1). The highest dark UV band (R_(F)=0.74) is scrapedfrom the plate and the product eluted with THF (yield: 98=100%).

Activation of carboxyl group with pentafluorophenol: 10 mg ofpentafluorophenol (PFP) in THF is added to cholesterol phthalatefollowed by 5 μl DIIC (diisopropylcarbodiimide). The solution is reacted1 hr at room temperature (RT) in a reaction vial, and the activatedcholesterol-phthalate-PFP is purified by TLC (CHCl₃/CH₃OH 30:5), andeluted with THF (yield: 100%).

Production of activated amide: Activated cholesterol-phthalate-PFP inTHF is evaporated to 10 μl and 2 equiv primary amine dissolved in DMF isadded. DMF can cause a side reaction but this is not a problem if excessamine is present. The addition of alcohols such as methanol or ethanolwill greatly reduce the yield. Three microliters DIIC are added and thesolution is reacted overnight at RT and purified by TLC (CHCl₃/CH₃OH30:5) (yield 98%). Importantly, only Silica Gel G-25 UV254 plates(Alltech 809021) were used for purification. Silica gel 60 platesproduce a fuzzy indistinct band that impairs purification.

Example 5 Synthesis of Adriamycin-Cholesterol (I)

Activated cholesterol-phthalate-PFP in THF is evaporated to 10 μl. 2equivalents of adriamycin dissolved in DMF is added. Side reactions withDMF may be reduced by using excess amine. The addition of alcohols suchas methanol or ethanol will greatly reduce the yield. Three microlitersDIIC are added and the solution is reacted overnight at RT and purifiedby TLC (CHCl₃/CH₃OH 30:5) (yield 95%). This band contains theadriamycin-DIIC conjugate and product. The cholesterol-adriamycinconjugate can be isolated by C₁₈-reverse-phase HPLC in 100% CH₃OH (flowrate=0.5 ml/min, detection=A₄₇₁). The product elutes at 4.7 min, abouthalfway between cholesterol and adriamycin. Overall yield: 95%. Thestructure of the adriamycin-cholesterol conjugate (I) is depicted inFIG. 5.

Example 6 Conjugation of Hydroxy-Containing Compounds with ¹⁴CCholesterol

N-[p-maleimidophenyl]isocyanate (PMPI)(5 mg) is mixed with 1 equiv. of ahydroxy-containing compound in 200 μl DMSO and reacted 30 min at roomtemperature. Thiocholesterol (6.4 mg) is added. The mixture is incubatedfor 120 min at room temperature and the product isolated by thin-layerchromatography using Silica Gel G UV 254 plates precoated with 0.1M EDTApH 8.0, with EtOH/H2O 1:1 as the solvent.

Example 7 Synthesis of Tetracycline-Cholesterol (II)

Tetracycline is lyophilized to remove any solvent and 2.2 mgtetracycline in 200 μl DMSO is mixed 5 mg PMPI and reacted 30 min atroom temperature. Thiocholesterol (6.4 mg) is added. The mixture isincubated for 120 min at room temperature and the product isolated bythin-layer chromatography using Silica Gel G UV 254 plates precoatedwith 0.1M EDTA pH 8.0, with EtOH/H2O 1:1 as the solvent. The structureof the tetracycline-cholesterol conjugate (II) is depicted in FIG. 6.

REFERENCES

Each of the reference listed below is incorporated herein by referencein its entirety.

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1. An artificial LDL particle comprising an outer phospholipid monolayerand a solid lipid core, wherein the outer phospholipid monolayercomprises at least one apolipoprotein and the solid lipid core containsat least one therapeutic agent.
 2. The artificial LDL particle of claim1, wherein the at least one apolipoprotein is ApoE.
 3. The artificialLDL particle of claim 2, wherein the at least one apolipoprotein isApoE3.
 4. The artificial LDL particle of claim 3, wherein the outerphospholipid monolayer further comprises one or more oxysterols and/oran additional apolipoprotein selected from the group consisting of ApoBand ApoE4.
 5. The artificial LDL particle of claim 1, wherein the atleast one therapeutic agent is selected from the group consisting of:amino acids, peptides, proteins, carbohydrates and lipids.
 6. Theartificial LDL particle of claim 1, wherein the at least one therapeuticagent is a conjugate formed between cholesterol and an agent selectedfrom the group consisting of: amino acids, peptides, proteins, nucleicacids, carbohydrates and lipids.
 7. The artificial LDL particle of claim5, wherein the therapeutic agent is selected from the group consistingof: neurotrophic factors, growth factors, enzymes, antibodies,neurotransmitters, neuromodulators, antibiotics, antiviral agents,antifungal agents and chemotherapeutic agents.
 8. The artificial LDLparticle of claim 6, wherein the therapeutic agent is selected from thegroup consisting of: neurotrophic factors, growth factors, enzymes,neurotransmitters, neuromodulators, antibiotics, antiviral agents,antifungal agents and chemotherapeutic agents.
 9. The artificial LDLparticle of claim 1, wherein the outer phospholipid monolayer comprisesphosphatidylcholine and at least one apolipoprotein.
 10. The artificialLDL particle of claim 9, wherein the at least one apolipoprotein isApoE.
 11. The artificial LDL particle of claim 1, wherein the particlehas a diameter between about 15 and 50 nm.
 12. The artificial LDLparticle of claim 1, wherein the particle has a diameter between about20 and 30 nm.
 13. The artificial LDL particle of claim 1, wherein theparticle has a density between about 1.00 and 1.07 g/ml.
 14. Theartificial LDL particle of claim 1, wherein the particle has a densitybetween about 1.02 and 1.06 g/ml.
 15. The artificial LDL particle ofclaim 1, wherein the particle has a serum stability of at least twohours.
 16. The artificial LDL particle of claim 1, wherein the particleis transported across the blood-brain barrier (BBB) by transcytosis. 17.The artificial LDL particle of claim 1, wherein the particle has atleast a 3-fold greater uptake specificity for brain compared to liver.18. The artificial LDL particle of claim 1, wherein the at least onetherapeutic agent is a conjugate formed between cholesterol andadriamycin.
 19. The artificial LDL particle of claim 1, wherein the atleast one therapeutic agent is a conjugate formed between cholesteroland tetracycline.
 20. The artificial LDL particle of claim 18, whereinthe cholesterol and adriamycin of the conjugate are linked by an esterbond.
 21. The artificial LDL particle of claim 19, wherein thecholesterol and tetracycline of the conjugate are linked by an esterbond.
 22. An artificial LDL particle for delivery of an agent across theblood-brain barrier comprising an outer phosphatidylcholine monolayer, asolid lipid core comprising fatty acyl-cholesterol esters, and ApoE inthe outer monolayer.
 23. The artificial LDL particle of claim 22,wherein the solid lipid core further comprises cholesterol.
 24. Theartificial LDL particle of claim 22, wherein the ApoE in the outermonolayer is ApoE3.
 25. A composition for delivery of an agent acrossthe blood-brain barrier comprising the artificial LDL particle of claim1 and a pharmaceutically acceptable carrier.
 26. A composition fordelivery of an agent across the blood-brain barrier comprising theartificial LDL particle of claim 4 and a pharmaceutically acceptablecarrier.
 27. A composition for delivery of an agent across theblood-brain barrier comprising the artificial LDL particle of claim 5and a pharmaceutically acceptable carrier.
 28. A conjugate comprisingcholesterol linked to a therapeutic agent selected from the groupconsisting of: amino acids, peptides, proteins, nucleic acids,carbohydrates and lipids.
 29. The conjugate of claim 28, wherein thetherapeutic agent is selected from the group consisting of: neurotrophicfactors, growth factors, enzymes, antibodies, neurotransmitters,neuromodulators, antibiotics, antiviral agents, antifungal agents andchemotherapeutic agents.
 30. The conjugate of claim 29, wherein thetherapeutic agent is adriamycin.
 31. The conjugate of claim 30, whereinthe adriamycin and cholesterol are linked by an ester linkage.
 32. Theconjugate of claim 29, wherein the therapeutic agent is tetracycline.33. The conjugate of claim 32, wherein the tetracycline and cholesterolare linked by an ester linkage.
 34. A method of producing an artificialLDL particle of claim 1 comprising the steps of: 1) suspendingphospholipids containing conjugated or unconjugated therapeutic agent ina buffer solution; 2) sonicating the solution to form the outerphospholipid monolayer and solid lipid core; and 3) adding a solutioncomprising at least one apolipoprotein, wherein the apolipoprotein isincorporated into the outer phospholipid monolayer.
 35. The method ofclaim 34, wherein the artificial LDL particles produced have a diameterbetween 10 and 50 nm.
 36. A method for delivery a substance across theblood-brain barrier, said method comprising administering an effectiveamount of the composition of claim 25 to a mammal in need thereof.
 37. Amethod for delivery a substance across the blood-brain barrier, saidmethod comprising administering an effective amount of the compositionof claim 26 to a mammal in need thereof.
 38. A method for delivery asubstance across the blood-brain barrier, said method comprisingadministering an effective amount of the composition of claim 27 to amammal in need thereof.
 39. A kit for delivering substances across theblood-brain barrier, said kit comprising a container containing thecomposition of claim 25 and instructions for use.
 40. A kit fordelivering substances across the blood-brain barrier, said kitcomprising a container containing the composition of claim 26 andinstructions for use.
 41. A kit for delivering substances across theblood-brain barrier, said kit comprising a container containing thecomposition of claim 27 and instructions for use.